Our laboratory has recently demonstrated that one of the major immediate early proteins of herpes simplex virus type 1 (HSV-1), ICP4, is localized within the tegument region of the virion (J. Virol. 63:3338, 1989). Other more recent data suggest that ICP0, but not ICP27, is also associated with purified virions. Related preliminary studies have shown that ICP4 is associated with the plasma membrane fraction of virus-infected cells. Based on the finding of ICP4 and ICP0 in purified virions, we suggest that it may be necessary to consider the possibility that ICP4 and ICP0 may act synergistically with the VP16 transactivating protein. The overall objective of the proposed research program is to define the characteristics as well as the role of the immediate early proteins represent a new direction of research concerning the HSV-1 immediate early proteins and based on the new direction of research concerning the HSV-1 immediate early proteins and based on the preliminary data available, we believe our laboratory is in a unique position to follow up on these studies. Experiments in the first three specific aims are designed to define and characterize the interaction of ICP4, ICP0 or ICP27 with the virion and the plasma membrane of the virus-infected cell. The fourth specific aim focuses on studies to identify the functional role of the virion-associated immediate early proteins. In the first specific aim, either purified proteins or synthetic peptides of ICP4, ICP0, and ICP27 will be used to prepare immunological reagents. Studies in the second specific aim will characterize the virion-associated ICP4 and ICP0 with regard to phosphorylation and possible fatty acid addition. In addition, use of chemical cross-linkers as well as nonsense and deletion mutants will be used to characterize further the association of ICP4 and ICP0 with the virion. In the third specific aim similar experiments will be used to characterize the interaction between the plasma membrane of HSV-1-infected cells and the immediate early proteins. Membrane-associated ICP4 will be examined relative to the degree of phosphorylation and the presence of fatty acyl groups. Nonsense and deletion mutants will be used to identify the domain of ICP4 responsible for its association with the plasma membrane. The fourth specific aim includes studies focused on defining the functional role of the virion- associated ICP4 and ICP0. Experiments will address the possibility that the virion-associated ICP4 and ICP0 function as transcriptional activators during the very early stages of the virus replication cycle. Studies are also proposed to determine if ICP4 and ICP0 represent structural components of the virion.